DE2822539B2 - Reagent system for the inhibition of adenylate kinase - Google Patents

Description

The invention relates to a Reagaizsystem for Inhibition of adenylate kinase and its use for determining creatine kinase activity

It has long been believed that creatine kinase activity is useful in the early diagnosis of Myoca ^ -dininfarkt (heart attack) and with progressive To determine muscular dystonia (J. Bkichem, 46, 103 [1959]; Rev. franc Ehid. Clin. BioL, 5,386 | .1960ft The bis now applied fundamental method was by Oliver in Biochem. J, 61, 116 (1955). This Method is based on the Creatine Lcinase CK enzyme catalyzed reaction, the catalysis being in the direction from a thermodynamic point of view The reaction in question can be represented as follows:

ATP + creatine

p H 9.0
pH 6.7

Creatine phosphate + ADP

(ATP-adenosine triphosphate, ADP = adenosine diphosphate).

This conversion is connected with my side reaction, which leads to substances which are photometrically let determine.

ATP + D-glucose HK = hexokinase

+ G-6-PD

D-glucose-6-phosphate + NADP

G-6-PD is glucose-6-phosphatdehyde; i'clrogenase and NADPH is nicotinamide-adenine-dinucotide-phosphate.

A major disadvantage of the above method is the fact that this system is affected by the activity of Adenylate kinase enzyme (AK), which is always present in normal sera and in particular, is influenced in hemolyzed samples and which is able to catalyze the formation of ATP according to the following scheme:

AK

2ADP

ΑΜΡ + ΑΓΡ,

wherein AMP adenosine-5'-monophospha); is

Accordingly, the presence of AK enzyme leads to an apparent increase in CreiHtinkinase (CK) activity.

Oliver himself observed that AK can be inhibited or inhibited by Ad «mosin-5'-monophosphate (AMP) at a concentration 5 to 10 times higher than that of ADP. Definitely inhibits at such concentrations AMP also affects the activity of CK and this effect is almost impossible to neglect. Various modifications have therefore been proposed by adding AMP to lower Concentrates are used so that there is no significant inhibition of CK, while at the same time there is sufficient, but not complete, inhibition of AK

This problem has also been worked on by other authors and other methoclein have been suggested To reduce the influence of AK activity, attempts have already been made to use AMP and diiadenosine pentaphosphate to use (Clin. Chem, 22, 1806 (1976]) or a fluoride (Clin. Chem, 22, 1078 [1976J, however, Diodenosine Pentaphosphate I-AMP does not lead to a satisfactory inhibition of AK from the

ADP + D-glucose-6-phosphate

6-phosphoglyconate + NADPH + H

Liver, while the fluoride at the concentration required to inhibit AK causes the precipitation of magnesium (which must be added to the system as an activator) in the form of MgF ₂

It was found according to the invention that it is possible to determine the creatine kinase activity without the above disadvantages, specifically with the aid of a reagent system consisting of AMP and a fluoride. In this way, the interfering activity of AK can be reduced to a negligible level, without any significant loss of creatine kinase activity and without precipitation of MgF ₂ . The reagent system according to the invention preferably contains the two reaction components in a ratio of 1: 0.15 to 1:25. Its use to determine the creatine kinase activity takes place in a volume ratio of

Serum to reagent system between 1:30 and 1:60.

It could be shown that the simultaneous exposure to low concentrations of AMP and Fluoride increases the performance of the system for determining creatine kinase activity

A strong inhibition of AK is achieved with simultaneous prevention of the inhibition of CK and the precipitation of magnesium fluoride. The fact is that when using AMP together with the Fluoride behaves differently towards AK

bo is determined in comparison to which one at the application can determine these two substances separately (Figs. 1, 2 and 4).

The inhibition or inhibition of AK is also extremely evident.

b5 In order to check the stability of the reagent system with regard to the precipitation of MgF ₂ , the reagent system was subjected to rapid temperature changes: It was removed from the refrigerator at 5 ° C

Immediately switched to a thermostat of 37 ° C and this freeze-thaw cycle and storage for 6 months at 5.20 and 37 ° C continuously. Under these conditions the enzymatic activity of HK and G-6-PD is obviously destroyed with the exception of the sample stored at 5 ° C. In any case, no precipitation of MgF ₂ took place at all

In addition, it should be noted that lead times (»Lag phases«) were never observed for more than 1 min with the method according to the invention and the one for it applied reaction system, which means that the CK activity has not been influenced in any way With usual reagents, the start-up time generally takes 3 to 5 minutes.

Creatine phosphate, ADP and AMP, HK (from yeast), G-6-PD (from L-mesenteroid), N-acetylcysts, D-glucose, Potassium fluoride, imidazole and acetic acid are available commercially.

Normal and abnormal sera (after myocardial infarction) and hemolysate of human erythrocytes were considered Source used for CK and AK. AK from the liver and muscles are obtained from the corresponding homogenates of the liver and muscles of rats and rabbits according to "Szasz et aL". The AK activity will according to Szasz et al, CHn. Chein, 22,1806,1976

Table I shows the concentrations of the reagents in the system according to the invention.

0.05 cm ^{3 of} serum were added to 3 cm ^{3 of the} reagent system, the latter being preheated to 37 ° C. The rate of reaction was recorded at 340 pm so that the run-up time of each serum could be determined. A Beckmann 25 spectrophotometer with a cuvette holder and thermostat was available. The reactin temperature for the determination of AK and CK was 37 ° C. The test results were confirmed by statistical analysis using Student's "t" test (Snedecor, C. W, and Cochran, W. G, Statistical Method, The Iowa State University Press , Ames, Iowa, 1968)

The inhibition of the AK activity and the CK activity by AMP and the fluoride was determined

Without fluoride, 95% inhibition of AK was achieved with more than 20 mmol / l AMP (FIG. 1). Without AMP 25 mmol / l were required for 95% inhibition Fluoride (Fig. 2). 20 mmol / 1 AMP also inhibited more than 10% of the CK activity and 25 mmol / 1 fluoride at least 4% of the CK activity (Fig. 3).

If AMP + fluoride is used according to the invention, this is achieved with just 2 mmol / 1 AMP and 6 mmol / 1 fluoride inhibited 95% of the AK activity (FIG. 4). Mixtures go from Fig. 5 AMP + fluoride for a certain percentage of inhibition of AK.

At the concentrations used, the inhibition of CK activity was no more than 3% (Fig. 6).

The method according to the invention was tested by determining the CK activity in normal sera without erythrocyte AK and with increasing amounts of erythrocyte AK. The samples were analyzed according to the method described and also using 5 mmol / 1 AMP. No significant differences were found between normal samples and those with additional erythrocyte AK in the method according to the invention, in which 2 mmol / 1 AMP and 6 mmol / 1 fluoride were used (P> 0.05).

ι ο

On the other hand, it was only AMP in an amount of

If 5 mmol / 1 is applied, a very significant difference is found (P <0ßl) and values were obtained for the samples with additional erythrocyte AK, which is the value *! of normal samples by about 50%.

In addition, the inhibition of AK from liver, heart or muscle with 2 mmol / 1 AMP and

6 mmol / 1 fluoride determined Table 2 shows the results.

The inhibition of the AK activity was always over 70%.

Table I.

Concentrations in the reaction mixture 100 mmol / l Imidazolactate buffer 3OmMoW Cratin phosphate 2 mmol / 1 ADP 2OmMoW N-acetylcysteine 20 mmol / l D-glucose 10 mmol / l Mg acetate 2 mmol / 1 NADP + 2000 U / l HK 2000 U / l G-6-PD pH 6.7 (25 ° Cl

During the tests, however, AMP and fluoride were used in different concentrations the optimal values ultimately used were 2 mmol / 1 AMP and 6 mmol / 1 fluoride.

Table II

Influence of 2 mmol / 1 AMP + 6 mmol / 1 fluoride on the adenylate kinase activity

AK off Szasc author. 2 mmol / 1 AMP % Inhi (10) + 6 mmol fluoride *) bation AK AK (mU / ml) (mU / ml) 76 Rats 72 -Liver 864 205 72 -Muscle 3 064 843 -Heart 1 755 483 72 Rabbits 80 -Liver 265 73 81 -Muscle 1 981 389 -Heart 989 189 79 Monkeys 83 -Liver 1 687 355 86 -Muscle 36 335 6154 -Heart 4,433 613 95 people 97 -Liver 1 133 58 96 -Muscle 7 802 245 96 -Heart 1 275 49 98 Erythrocytes 978 40 Tile 285 7th

*) The indicated concentrations of AMP and fluoride were the reagent for the determination of the AK according to the Comparative method 10 added.

The abbreviations used here have the following meanings:

CK = creatine kinase (EC 2.73.2.)

ATPiCreatine Phosphotransferase;
HK - hexokinase (EC 2.7.1.1.)

ATP: D-hexose-6-phosphotransferase;
AK = adenylate kinase (myokinase) (EC 2.7.43.)

ATP: AMP phosphotransferase;
G-6-PD = glucose-δ-phosphate dehydrogenase

(EC 1.1.1.49)

D-glucose-6-phosphate-NADP + -

1 -oxidoreductase.

The following should be noted about the diagrams:
F i g.! shows the. Influence of AMP on the activity of adenylate kinase from erythrocytes% AK inhibition is plotted on the ordinate and the concentrations of AMP in mmol / 1 are plotted on the abscissa. The following hemoglobin concentrations were examined:

- O-225 mg / dl
- Δ - 50 mg / dl

- O-110 mg / ml

- χ - 25 mg / ml

F i g. 2 shows the influence of the fluoride on the activity of adenylate kinase. On the ordinate is the % Inhibition of AK and the concentrations of fluoride in mmol / 1 on the abscissa. The hemoglobin concentration was 225 mg / dl.

Fig.3 shows the influence of AMP + fluoride on the Activity of serum creatine kinase. All values are mean values from 5 determinations. All CK activities were corrected taking AK activity into account

The CK activities were between 196 and 1030 U / l.

! ■>

The same samples were used for AMP and fluoride. The ordinate shows Vo inhibition for creatine kinase and the abscissa shows the concentrations for AMP and fluoride in mmol / l.

Fig.4 shows the influence of AMP + fluoride on the Activity of adenylate kinase from erythrocytes. The hemoglobin concentration was 225 mg / dl.

AMP concentrations:

- Ο— OmMol / l

- χ— 5 mmol / 1
—O— 2 mmol / 1
- Δ - I mMoi / i
- D - IO mmol / 1

The% inhibition of AK and is on the ordinate the fluoride concentration in mmol / 1 is plotted on the abscissa.

Fig.5 shows the influence of AMP + fluoride on the Activity of adenylate kinase from erythrocytes. The three curves relate to an AK inhibition of:

-O-

95%

- Π— 90%
—Δ— 80%

The fluoride concentration is indicated on the ordinate and the AMP concentration is indicated on the abscissa.

F i g. 6 shows the influence of fluoride on the activity of creatine kinase at constant AMP concentration of 2 mmol / 1. The ordinate is% inhibition (relative) of CK and the abscissa is the fluoride concentration given in mmol / 1.

For this purpose 3 sheets of drawings

Claims (3)

Patent claims: 1. Reagent system for inhibition: of adenylate kinase, consisting of adenosine-5 'monophosphate and a fluoride. 2. Reagent system according to claim 1, characterized in that the two reaction components in a ratio of 1: 0.15 to 1:25 are included. 3. Use of the reagent system according to claim 1 or 2 for determining the creatine kinase activity in a volume ratio of serum to reagent system between 1:30 and 1:60.